Many methods are known for detecting analytes of various kinds using a reactive solid surface. In many hybridization assay formats, for example, a label is detected on the surface of a substrate, e.g., on a glass or plastic bead, plate, tube or the like, to indicate the presence and/or quantity of an analyte of interest. There are also many types of chromatographic procedures in which reactive surfaces are used to facilitate the separation and/or detection of different types of analyte molecules. In still another context, mass biosensors are used to measure microquantities of biological materials, and, as with the aforementioned contexts, involve the use of a modified surface which selectively binds a particular component. Although the present invention is adaptable to a wide variety of contexts, it is particularly suited to use in conjunction with such mass biosensors.
As explained in commonly assigned U.S. Pat. No. 5,130,257 to Baer et al., European Patent Publication No. 416,730, inventors Tom-Moy et al., and co-pending U.S. Patent application Ser. No. 08/041,662, filed Apr. 1, 1993 (entitled "A Mass Sensor for Measuring Analytes in a Sample," inventors C. A. Myerholtz et al.), a preferred type of mass biosensor uses a piezoelectric crystal as an acoustic waveguide. Selective mass detection with such devices is achieved by coating the surface of the device with a chemically reactive layer that preferentially reacts with the substance to be detected such that the mass present on the reactive layer changes proportionately, i.e., relative to the amount of the substance to be detected. These devices thus function as chemical sensors that can measure the concentration of analytes in a solution into which the detector is immersed. For example, and as explained in U.S. Patent application Ser. No. 08/041,662, cited above, piezoelectric surface wave devices have been used to measure the concentration of a specific antibody in solution using a conventional assay format, as follows. The mass-sensitive surface of the device is coated with a receptor layer which contains the antigen corresponding to the antibody. The device is then exposed to a sample solution, and antibody present in the solution will bind to the surface of the device, thereby increasing the mass loading of the upper surface. An input transducer generates a periodic acoustic wave from a periodic electrical input signal. Radio frequency energy coupled into the device through the input transducer is converted to a surface acoustic wave confined to within a few wavelengths of the surface. The velocity of the surface acoustic wave will vary according to the mass loading on the top surface of the device. The surface acoustic wave propagates along the surface of the device until it encounters the output transducer, which converts the surface acoustic wave back into radio frequency energy. The change in propagation velocity of the surface acoustic wave corresponds to the mass bound to the surface of the crystal. By monitoring the frequency or relative phases of the input and output electrical signals, the mass changes at the surface of the crystal can be measured. Such acoustic waveguide devices can utilize different wave motions, including surface transverse waves (STWs), Rayleigh waves (SAWs), Lamb waves, and surface-skimming bulk waves (SSBWs), although STW devices are preferred.
The present invention makes use of the strong interaction between biotin and a biotin-binding protein to bind analyte molecules to the surface of a substrate, such as the surface of a piezoelectric crystal in a surface transverse wave biosensor. The use of the extremely high affinity (K.sub.a .apprxeq.10.sup.15 M.sup.-1), although noncovalent, bond formed between biotin and the biotin-binding protein avidin has been well-documented. M. Wilchek et al., in "The Avidin-Biotin Complex in Bioanalytical Applications," Anal. Biochem. 171:1-32 (1988), present an overview of a number of contexts within which the avidin-biotin complex has proven useful. There are additional references which propose the use of the avidin-biotin interaction in binding materials to surfaces. PCT Publication No. WO91/07087, for example, describes a technique for creating regions on a solid surface which are capable of selectively immobilizing an "anti-ligand" through biotin-avidin complexation. U.S. Pat. No. 4,952,519 and European Patent Publication No. 396,116 relate to the derivatization of the surface of a solid support so as to bind biotin or avidin thereto; PCT Publication No. WO88/04777 also describes an analyte detecting device containing a detection surface on which avidin or biotin is immobilized, while U.S. Pat. No. 4,478,914 and U.S. RE31,712, both to Giese, describe a modified surface coated with alternating layers of a ligand-binding protein such as avidin and a reactive ligand extender such as biotin. Commonly assigned European Patent Publication No. 416,730, cited previously, describes a mass biosensor in which a ligand-binding layer such as an avidin coating is provided on the piezoelectric crystal surface of the device, on top of which is provided a ligand-bearing coating such as a layer of biotinylated antibody.
Although a number of references thus describe the use of biotin-avidin complexation in a variety of analyte detection and quantitation procedures, none provide a method for attaching low molecular weight analytes--such as environmental analytes of interest--to a solid phase surface using biotin-avidin complexation. Typically, as noted above, biotin has been attached to large molecules such as protein and nucleic acid moieties. It can be difficult to adsorb small analytes, or to bind small analytes covalently, to the surfaces of plates, tubes, or the like. A strong, preferably covalent, attachment of low molecular weight moieties is particularly important with surface transverse wave devices, so that the device can be used repetitively without the bound moieties being washed away between individual cycles. The method of the invention addresses this need in the art and provides a simple, reliable method of attaching small analyte molecules to substrate surfaces, such as the mass-sensitive surfaces of piezoelectric surface transverse wave devices.